Polypeptide binding to human syntaxin 1a

ABSTRACT

There is provided a polypeptide that binds to syntaxin-1a and comprises the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing, or a polypeptide having a homology of at least approximately 90% to the amino acid sequence that binds to syntaxin-1a, a partial peptide thereof, and a polynucleotide encoding any of these polypeptides or the complementary strand thereof, as well as an inhibitor, an antagonist, and an activator of physiological activity of any of the polypeptides. Further, a pharmaceutical composition that utilizes these is provided. There is also provided a method for producing the polypeptides or the peptide by genetic engineering techniques, a method for identifying the inhibitor, antagonist, or activator, and a method and a kit for diagnosing a disease with which the polypeptides are associated.

TECHNICAL FIELD

The present invention relates to a polypeptide that has avesicle-associated membrane protein (hereinafter also referred to as“VAMP”)-homologous region and binds to syntaxin-1a, as well as a peptidehaving a part of the polypeptide. The present invention also relates toa polypeptide or peptide that promotes the formation of a complexcomprising human syntaxin-1a, 25 kDa synaptosomal-associated protein(hereinafter also referred to as “SNAP-25”) and synaptotagmin, whereinthe complex participates in the fusion of synaptic vesicles withpresynaptic membranes. The present invention further relates to aregulator of exocytosis that results from the fusion of synapticvesicles with presynaptic membranes, wherein the regulator contains thepolypeptide or the peptide and acts by a binding function to humansyntaxin-1a. The present invention also relates to the following: apolynucleotide comprising a nucleotide sequence encoding the polypeptideor peptide, or the complementary sequence thereof; a recombinant vectorcontaining the polynucleotide; or a transformant into which therecombinant vector is transfected. The present invention further relatesto the following: an antibody against the polypeptide or peptide; acompound having an ability to interact with the polypeptide, peptide, orthe polynucleotide; a pharmaceutical composition containing one or moreof these substances; or a pharmaceutical composition that has an actionto control the release of a neurotransmitter.

The present invention further relates to a method for controlling therelease of a neurotransmitter by controlling the binding of thepolypeptide to human syntaxin-1a, and to a method for preventing and/ortreating diseases that are caused by the abnormal release ofneurotransmitters. The present invention still further relates to ameasurement means for diagnosis that comprises measuring the polypeptideor peptide or the polynucleotide, a method for producing the polypeptideor peptide using the transformant or the recombinant vector, a methodfor identifying a compound that acts on the binding of the polypeptideto human syntaxin-1a or a compound that regulates expression of thepolynucleotide, and a reagent kit to be used with the measurement meansand the identification method.

BACKGROUND ART

Syntaxin-1, which is mainly present in the brain, is a protein thatrelates to a release mechanism for neurotransmitters. The isoforms,syntaxin 1a and 1b, are known. Upon release of neurotransmitters(exocytosis), syntaxin 1 forms a complex with synaptosomal-associated 25kDa protein (SNAP-25), vesicle-associated membrane protein (VAMP, alsocalled “synaptobrevin”) and synaptotagmin/p65, and participates in theprocess of docking and/or fusion of synaptic vesicles with presynapticmembranes. In the complex, VAMP is localized in synaptic vesicles andfunctions as a v-SNARE (vesicle-soluble N-ethylmaleimide-sensitivefactor attachment receptor), and syntaxin 1 and SNAP-25 are mainlylocalized in presynaptic membranes and function as a t-SNARE(target-soluble N-ethylmaleimide-sensitive factor attachment receptor).SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)formation is inhibited by MUNC-18, which binds to syntaxin 1.

The binding of syntaxin 1a, which is a principal molecule among thesyntaxins, to m-tomosyn may be significant in the following respects.Among the many proteins involved in docking and membrane fusion ofsynaptic vesicles upon exocytosis of neurotransmitters, syntaxin is at-SNARE that is considered to be the molecule fulfilling the main roletherein. Syntaxin 1 combines with SNAP-25, which is another t-SNARE, aswell as with VAMP and synaptotagmin, which are two kinds of v-SNARE, toform a 7S (S: sedimentation coefficient) complex, and consequently thesynaptotagmin therein is replaced by α-SNAP (soluble NSF-attachmentprotein) and NSF (N-ethylmaleimide-sensitive factor) to form a 20Scomplex, which is considered to be extremely important for the start ofthe docking and membrane fusion mentioned above.

Fujita et al. analyzed the glycerol gradient fractions of rat brain LP2fraction, using antibodies against each constitutive protein of SNARE(such as anti-tomosyn antibody), for the constitutive proteins in eachfraction. Their findings revealed that m-tomosyn forms a 10S complexwith SNAP-25 and synaptotagmin. They also exhibited data suggesting thatm-tomosyn dissociates MUNC-18 from a complex comprising syntaxin andMUNC-18 to bind to the syntaxin (non-patent document 1). Further, highlevel expression of m-tomosyn in a PC12 cell reduced the amount ofcalcium-dependent exocytosis by 30% (non-patent document 1). MUNC-18 andsynaptophysin are considered to suppressively control the formation ofSNARE by binding to syntaxin and VAMP, respectively. It is believed thatm-tomosyn replaces MUNC-18 to bind to syntaxin-1a due to its having ahigher affinity than MUNC-18, and thereby forms a 10S complex, whichthen sequentially changes to a 7S complex and then a 20S complex throughsteps of some kind, and finally resulting in the fusion of synapticvesicles with presynaptic membranes.

In short, there is a possibility that m-tomosyn, which is a proteinhaving high binding affinity to syntaxin that is believed to play acentral role in the fusion of synaptic vesicle with presynapticmembrane, plays a highly important role in the docking and fusion ofsynaptic vesicles.

The references cited herein are listed below.

Patent document 1: Method for producing recombinant baculovirusexpression vector; Japanese Patent No. 2129487.

Patent document 2: Method for producing polypeptide; Japanese PatentNo.2644447.

Non-patent document 1: Fujita, Y. et al., Tomosyn: a syntaxin-1-bindingprotein that forms a novel complex in the neurotransmitter releaseprocess; Neuron, (1998) 20, p.905-915.

Non-patent document 2: Yokoyama, S. et al., Three splicing variants oftomosyn and identification of their syntaxin-binding region; Biochemicaland Biophysical Research Communications, (1999) 256, p.218-222.

Non-patent document 3: Masuda, E. S. et al., Tomosyn binds t-SNAREproteins via a VAMP-like coiled coil; Neuron, (1998) 21, p.479-480.

Non patent document 4: Lehman, K. et al., Yeast homologue of tomosyn andLethal giant larvae function in exocytosis and are associated with theplasma membrane SNARE; Sec. 9, Journal of Cell Biology, (1999) 146,p.125-140.

Non-patent document 5: Sambrook et al., “Molecular Cloning, A LaboratoryManual, second edition” Cold Spring Harbor Laboratory (1989).

Non-patent document 6: Masami Muramatu, “Lab Manual of GeneticEngineering”, MARUZEN Co., Ltd. (1988).

Non-patent document 7: Ehrlich, H. E et al., “PCR Technology, Theory andApplication of DNA Amplification,” Stockton Press (1989).

Non-patent document 8: Ulmer, K. M., Science, (1983) 219, p.666-671.

Non-patent document 9: Nature, (1957) 179, p.160-161.

Non-patent document 10: Hata, Y. et al., Synaptic vesicle fusion complexcontains unc-18 homologue bound to syntaxin (1993), Nature, (1993) 366,p.347-351.

Non-patent document 11: Hata, Y. et al., A novel ubiquitous form ofMunc-18 interacts with multiple syntaxins, Journal of BiologicalChemistry, (1995) 270, pp.13022-13028.

Non-patent document 12: Kee, Y. et al., Distinct domains of syntaxin arerequired for synaptic vesicle complex formation and dissociation,Neuron, (1995) 14, p.991-998.

Non-patent document 13: Kee, Y. et al., Localization ofsynaptotagmin-binding domain on syntaxin, Journal of Neuroscience,(1996) 16, p.1975-1981.

DISCLOSURE OF THE INVENTION

In consideration of the facts described above, it is an object of thepresent invention to find and provide a polypeptide or peptide thatbinds to human syntaxin-1a. Another object of the present invention isto provide a polynucleotide encoding the polypeptide or peptide thatbinds to human syntaxin-1a and to provide a method for producing thepolypeptide or peptide that binds to human syntaxin- 1a using geneticengineering techniques. A further object of the present invention is toprovide an antibody for the polypeptide or peptide that binds to humansyntaxin-1a, and an inhibiter or a stabilizer of the binding of humansyntaxin-1a with the polypeptide or peptide that binds to humansyntaxin-1a. A still further object of the present invention is toidentify, using the aforementioned method and substances, an antibodyfor the polypeptide or peptide that binds to human syntaxin-1a, as wellas an inhibitor or stabilizer of the binding of human syntaxin-1a to apolypeptide or peptide that binds to human syntaxin-1a, and to provide apharmaceutical composition using the aforementioned substances, as wellas a measurement method and a reagent kit for diagnosis.

The present inventors have conducted concentrated studies to achieve theobjects mentioned above, and found that a part of the KIAA1006 clone(GenBank accession number: AB023223) that is disclosed in the human longchain cDNA analysis information database of the Kazusa DNA ResearchInstitute has high homology to the VAMP homologous region of ratm-tomosyn. More specifically, they performed a homology search in thehuman long chain cDNA analysis information database of the Kazusa DNAResearch Institute with respect to the VAMP homologous region that isindicated as being important in binding to syntaxin-1a in rat m-tomosyn,and selected KIAA 1006. The present inventors then determined thefull-length open reading frame (ORF) of KIAA1006 gene and carried outthe expression of the gene in a gene expression system using an insectcell to obtain a polypeptide that is the gene product encoded by thegene. Further, they demonstrated that the thus obtained polypeptidebinds to human syntaxin-1a. They completed the present invention byproviding a pharmaceutical composition for a disease attributable to thepolypeptide by regulating the function and physiological effects of thepolypeptide, as well as a measurement means for diagnosing the disease.

More specifically, the first aspect of the present invention relates toa polypeptide that binds to human syntaxin-1a, wherein the polypeptideis selected from the group consisting of:

(i) a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 in the Sequence Listing;

(ii) a polypeptide containing the polypeptide of the above (i);

(iii) a polypeptide having at least 90% homology to the amino acidsequence of the polypeptide of the above (i); and

(iv) a polypeptide having a mutation such as a deletion, substitution,addition or insertion of one or several amino acids with respect to theamino acid sequence of any of the polypeptides of the above (i) to(iii).

Another aspect of the present invention relates to a peptide comprisingat least five consecutive amino acid residues in the amino acid sequenceshown in SEQ ID NO: 1 in the Sequence Listing.

A further aspect of the present invention relates to the aforementionedpolypeptide or peptide, wherein the polypeptide or the peptide promotesthe formation of a complex that comprises human syntaxin-1a,synaptosomal-associated 25 kDa protein and synaptotagmin, and isinvolved in the fusion of a synaptic vesicle with a presynapticmembrane.

A further aspect of the present invention relates to a regulator ofexocytosis through fusion of a synaptic vesicle with a presynapticmembrane, wherein the regulator comprises the aforementioned polypeptideor peptide and has a binding function to human syntaxin-1a.

A still further aspect of the present invention relates to apolynucleotide comprising a nucleotide sequence encoding theaforementioned polypeptide or peptide, or a complementary sequencethereof.

A further aspect of the present invention relates to a polynucleotidethat hybridizes to the aforementioned polynucleotide under stringentconditions.

A still further aspect of the present invention relates to apolynucleotide having at least 15 consecutive nucleotides of theaforementioned polynucleotide.

A further aspect of the present invention relates to a recombinantvector containing the aforementioned polynucleotide.

A further aspect of the present invention relates to the aforementionedrecombinant vector, wherein the recombinant vector is a recombinantexpression vector.

A still further aspect of the present invention relates to atransformant that is transformed with the aforementioned recombinantvector.

A further aspect of the present invention relates to a method forproducing the aforementioned polypeptide or peptide, wherein the methodcomprises a step of incubating the aforementioned transformanttransformed with the aforementioned recombinant vector.

A still further aspect of the present invention relates to an antibodythat immunologically recognizes the aforementioned polypeptide orpeptide.

A further aspect of the present invention relates to the aforementionedantibody that inhibits the binding of the aforementioned polypeptide orpeptide to a protein that interacts with the polypeptide or the peptide.

A further aspect of the present invention relates to the aforementionedantibody, which inhibits the binding of the aforementioned polypeptideor peptide to human syntaxin-1a.

A further aspect of the present invention relates to a method foridentifying a compound that inhibits or stabilizes the binding of theaforementioned polypeptide or peptide to a protein that interacts withthe polypeptide or the peptide and/or a compound that inhibits orpromotes expression of the aforementioned polynucleotide, wherein themethod uses at least one member selected from the group consisting ofthe aforementioned polypeptide or peptide, the aforementionedpolynucleotide, the aforementioned vector, the aforementionedtransformant and the aforementioned antibody.

A still further aspect of the present invention relates to a method foridentifying a compound that inhibits or stabilizes the binding of theaforementioned polypeptide or peptide to a protein that interacts withthe polypeptide or peptide, wherein the method comprises steps of:contacting a test substance with the polypeptide or the peptide and aprotein that interacts with the polypeptide or peptide, under conditionsthat allow the binding of the polypeptide or the peptide to a proteinthat interacts with the polypeptide or peptide; and determining whetherthe test substance inhibits or stabilizes the binding of the polypeptideor the peptide to a protein that interacts with the polypeptide orpeptide, by detecting the presence, absence, or change of a signalgenerated by binding of the polypeptide or the peptide to a protein thatinteracts with the polypeptide or peptide.

A further aspect of the present invention relates to a method foridentifying a compound that inhibits or stabilizes the binding of theaforementioned polypeptide or peptide to human syntaxin-1a, wherein themethod comprises steps of: contacting a test substance with thepolypeptide or the peptide and human syntaxin 1a, under conditions thatallow the binding of the polypeptide or the peptide to humansyntaxin-1a; and determining whether the test substance inhibits orstabilizes the binding of the polypeptide or the peptide to humansyntaxin-1a, by detecting the presence, absence, or change of a signalgenerated by the binding of the polypeptide or the peptide to humansyntaxin-1a.

A still further aspect of the present invention relates to a method foridentifying a compound that inhibits or promotes expression of any ofthe aforementioned polynucleotides, wherein the method comprises stepsof: contacting a test substance with the polynucleotide under conditionsthat allow expression of the polynucleotide; and determining whether thetest substance inhibits or promotes expression of the polynucleotide bydetecting the presence, absence, or change of a signal generated byexpression of the polynucleotide.

A further aspect of the present invention relates to a compound that isidentified by any of the aforementioned method for identifying acompound.

A further aspect of the present invention relates to a compound thatinhibits or stabilizes the binding of the aforementioned polypeptide orpeptide to human syntaxin-1a by interacting with the polypeptide orpeptide.

A still further aspect of the present invention relates to a compoundthat inhibits or promotes expression of any of the aforementionedpolynucleotides by interacting with the polynucleotide.

A further aspect of the present invention relates to a pharmaceuticalcomposition comprising at least one member selected from the groupconsisting of the aforementioned polypeptide or peptide, theaforementioned polynucleotide, the aforementioned vector, theaforementioned transformant, the aforementioned antibody, and theaforementioned compound.

A further aspect of the present invention relates to the aforementionedpharmaceutical composition, which has a function to promote or inhibitthe release of neurotransmitters.

A further aspect of the present invention relates to the aforementionedpharmaceutical composition, which is used as a preventive and/ortherapeutic agent for a disease caused by an abnormal amount ofneurotransmitters.

A still further aspect of the present invention relates to a method forpromoting or inhibiting the release of neurotransmitters, wherein themethod comprises inhibiting or stabilizing the binding of theaforementioned polypeptide to human syntaxin-1a.

A further aspect of the present invention relates to a method forpreventing or inhibiting a disease caused by an abnormal amount ofneurotransmitters, wherein the method comprises inhibiting orstabilizing the binding of the aforementioned polypeptide to humansyntaxin-1a.

A further aspect of the present invention relates to a method formeasurement used in diagnosis of a disease caused by an abnormality inthe expression or function of the aforementioned polypeptide, whereinthe method comprises conducting an analysis employing (A) a nucleic acidencoding the polypeptide, and/or (B) the polypeptide as a marker.

A further aspect of the present invention relates to a method formeasurement used in diagnosis of a disease associated with an abnormalfunction of neurotransmittion caused by an abnormal amount of theaforementioned polypeptide, wherein the method comprises conducting ananalysis employing (A) a nucleic acid encoding the polypeptide and/or(B) the polypeptide as a marker.

A still further aspect of the present invention relates to a reagent kitcomprising at least one member selected from the group consisting of theaforementioned polypeptide or peptide, the aforementioned polynucleotideand the aforementioned antibody.

A further aspect of the present invention relates to a reagent kit foruse in the aforementioned identification method or the aforementionedmeasurement method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram to illustrate that synaptic vesicles fusewith presynaptic membrane in the exocytosis of neurotransmitters throughthe steps of: 1) replacement of MUNC-18 that is bound to syntaxin withtomosyn, 2) formation of a 10S complex, 3) formation of a 7S complex,and 4) formation of a 20S complex, in this order. The meaning of theeach symbol used in FIG. 1 is listed below.

PM: presynaptic membrane

SVM: synaptic vesicle membrane

Sy: syntaxin

M: MUNC-18

T: tomosyn

N: NSF

S: α-SNAP

25: SNAP-25

V: VAMP

St: synaptotagmin

FIG. 2 shows the binding of the KIAA1006 polypeptide and C-terminaltruncated KIAA1006 to syntaxin-1a. The polypeptide of the presentinvention and C-terminal truncated product, which were expressed andpurified according to a method described in Example herein, weresubjected to treatment with glutathione S-transferase (GST)-bindingglutathione-Sepharose and then syntaxin-1a-GST-bindingglutathione-Sepharose according to a method described in the Examplehereunder. Fractions that bound to GST-binding glutathione-Sepharose andsyntaxin-1a-GST-binding glutathione-Sepharose were eluted with a samplebuffer for sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE), and then analyzed together with a fraction unadsorbed to theGST-binding glutathione-Sepharose by Western blotting using anti-pentahistidine (penta His) antibody (Qiagen). In the figure, lanes 1 and 2show GST-adsorbed fractions, lanes 3 and 5 show GST-unadsorbedfractions, and lanes 4 and 6 show syntaxin-1a-GST-adsorbed fractions,wherein lanes 1, 3 and 4 show the KIAA1006 polypeptide (1-1186AA), andlanes 2, 5 and 6 show the C-terminal truncated product (1-1157AA) ofKIAA1006 polypeptide.

DETAILED DESCRIPTION OF INVENTION

The present invention claims the benefit of priority of Japanese PatentApplication No. 2002-75551, which is incorporated herein by reference.

Technical and scientific terms used herein have the meanings asunderstood generally by those skilled in the art, unless otherwisedefined. Reference is made herein to a variety of methods that arewell-known to those skilled in the art. Data from publications and thelike that discloses such cited well-known methods are deemed completelyincorporated herein in their entirety by reference.

A mode of embodiment of the present invention may be described in moredetail hereafter. The following detailed description is illustrative andmerely explanatory, and it does not limit the scope of the presentinvention.

(Polypeptide or Peptide)

A polypeptide provided according to the present invention is apolypeptide encoded by KIAA1006, which is a gene selected from a longchain cDNA library derived from the human brain (disclosed by the KazusaDNA Research Institute) by carrying out a homology search for the VAMPhomologous region that has been indicated as being involved in thebinding of rat m-tomosyn to syntaxin-1a (non-patent document 2 andnon-patent document 3). The polypeptide is obtained as a soluble proteinby expressing the ORF region of KIAA1006 in a gene expression systemusing baculovirus and an insect cell, sf9. The full-length ORF of theKIAA1006 gene comprises 3,558 base pairs, and the product of the genecomprises 1,186 amino acid residues. The homology of KIAA1006 to ratm-tomosyn is 57.6% at the nucleotide level and 67.9% at the amino acidlevel. In the present invention, it was revealed that the polypeptidebinds to human syntaxin-1a. Hereinunder, the ORF region of KIAA1006 isalso referred to as “KIAA1006 polypeptide.”

As described above, KIAA1006 polypeptide exhibits a high homology to ratm-tomosyn, so it is considered to function in a similar manner tom-tomosyn in humans.

In the exocytosis of a neurotransmitter, synaptic vesicles fuse with thepresynaptic membrane through the following steps of: 1) replacement ofMUNC-18 binding to syntaxin, with tomosyn; 2) formation of a 10Scomplex; 3) formation of a 7S complex; and 4) formation of a 20Scomplex; in this order (refer to FIG. 1) (non-patent document 2 andnon-patent document 3). The term “promotion of complex formation” mayrefer to promotion of 10S complex formation or promotion of 7S complexformation, and may also refer to promotion of 20S complex formation. Inthe process of these complex formations, m-tomosyn is the protein havingthe highest affinity to syntaxin among the proteins that bind tosyntaxin, and therefore plays a central role in the exocytosis ofneurotransmitters. It is considered that the polypeptide and peptide ofthe present invention have an action that promotes the formation ofthese complexes similarly to m-tomosyn.

High level expression of m-tomosyn in PC12 cells inhibitscalcium-dependent exocytosis by 30% (non-patent document 1). Exocytosisis also inhibited in yeast strains with a deleted homolog of m-tomosyn,which results in inhibition of proliferation (non-patent document 4).These facts show that m-tomosyn plays a regulating role in this step. Inother words, overexpressed tomosyn generates an inhibitory effect on 10Scomplex formation. Further, when tomosyn is not present, the step doesnot progress, which results in down-regulation of exocytosis as in thecase where tomosyn is overexpressed. It is considered that thepolypeptide and peptide of the present invention have a function toregulate this step by binding to syntaxin in a similar manner tom-tomosyn.

It is disclosed and reported in the aforementioned database thatKIAA1006 is specifically expressed in the brain. Therefore, it isbelieved that the KIAA1006 polypeptide is an important factor in humans,as m-tomosyn in rats, to regulate the docking and fusion of synapticvesicles through syntaxin.

The polypeptide of the present invention can be obtained by knowngenetic engineering techniques based on the nucleotide sequenceinformation of KIAA1006 cDNA (GenBank accession number: AB023223). Forexample, the polypeptide can be obtained by amplifying the gene encodingthe ORF region of KIAA1006 gene by the polymerase chain reaction (PCR)using a plasmid hk10084 that contains KIAA1006 which was purchased fromKazusa DNA Research Institute, as a template, and then infecting aninsect cell, Sf9, with a baculovirus integrated with the gene to induceexpression, as described above or in the hereinafter-described example.Further, it is possible to obtain the polypeptide of the presentinvention by, for example, amplifying the gene of interest using a humanbrain derived cDNA library as a template and a suitable primer designedby a known method based on the aforementioned KIAA1006 cDNA nucleotidesequence information, and expressing the resultant gene by a knowngenetic engineering technique.

As used herein, the term “polypeptide” refers to a long chain peptidesuch as a protein containing two or more amino acids that are bound toone another by a peptide bond or a modified peptide bond. A short chainpeptide, which is also called an “oligopeptide” or “oligomer”, isreferred to as only a “peptide”.

The polypeptide of the present invention is a polypeptide having afunction to bind to human syntaxin-1a (syntaxin-1a) and comprises theamino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.

The polypeptide of the present invention may be a polypeptide that bindsto human syntaxin-1a (syntaxin-1a) and contains a polypeptide comprisingthe amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.

The polypeptide of the present invention may also be a polypeptide thatbinds to human syntaxin-1a (syntaxin-1a) and has a homology of 90% ormore to the polypeptide shown in SEQ ID NO: 1 in the Sequence Listing atan amino acid sequence level. Techniques for determining the homology ofamino acid sequences are known. Examples of useful methods include amethod in which an amino acid sequence is determined directly or amethod in which a nucleotide sequence of cDNA is first determined andthe amino acid sequence encoded thereby is then deduced. It is possibleto determine the ability of binding to human syntaxin-1a (syntaxin-1a)by the method described hereinafter in the Example.

Further, based on the thus specified polypeptide, a polypeptidecomprising an amino acid sequence having a mutation such as a deletion,substitution, addition or insertion of one amino acid or more, forexample, 1 to 100, preferably 1 to 30, more preferably 1 to 20, furtherpreferably 1 to 10, and particularly preferably one or several aminoacids, is also provided by referring to the ability of binding to humansyntaxin-1a. A polypeptide having such mutation may be a naturallyexisting polypeptide or a polypeptide in which a mutation is introduced.Methods for introducing a mutation are well known, for example, methodsinvolving site-specific mutagenesis, genetic homologous recombination,primer extension, polymerase chain reaction (PCR) or the like can beused alone or in suitable combination. The above methods can be carriedout, for example, according to a method shown in the publications(non-patent document 5, non-patent document 6 and non-patent document 7)or by modified methods thereof. The Ulmer's techniques (non-patentdocument 8) may also be utilized.

When introducing a mutation as described above, in view of avoiding achange in the fundamental properties (such as physical properties,function, physiological activity, and immunological activity) of thepolypeptide, mutual substitution among homologous amino acids (polaramino acids, non-polar amino acids, hydrophobic amino acids, hydrophilicamino acids, positively-charged amino acids, negatively-charged aminoacids and aromatic amino acids or the like) may be readily conceived. Inaddition, these available peptides can be modified to the extent that nosignificant functional change is involved, such as modification of theirconstituent amino group or carboxyl group or the like by an amidation orthe like.

The peptide of the present invention includes a peptide containing apartial sequence of the polypeptide shown in SEQ ID NO: 1 in theSequence Listing. The peptide preferably comprises 5 or more, morepreferably 8 or more, further preferably 12 or more, and particularlypreferably 15 or more consecutive amino acids as its minimum unit. Thesepeptides can be produced using a known method for producing a peptide inaccordance to their amino acid sequence information. Examples of knownmethods for chemical synthesis of a peptide include solid phasesynthesis, liquid phase synthesis and the like, and any method thereofcan be used herein. Alternatively, the peptide can be obtained by thesame method as that for the polypeptide mentioned above using geneticengineering techniques. The peptide can be also obtained by cleaving thepolypeptide of the present invention using a suitable peptidase.

A peptide obtained by these methods can be used as a reagent or astandard substance or the like. Among these peptides, a peptide thatbinds to syntaxin-1a is useful as a substance that inhibits the bindingof the polypeptide of the present invention to syntaxin-1a, and is alsouseful for screening for a substance that regulates the activity of thepolypeptide of the present invention. A peptide that is immunologicallyrecognized, such as, for example, an epitope peptide, can be used forproducing an antibody that is specific to the polypeptide of the presentinvention, as an antigen by itself or by binding it to a carrier (e.g.,keyhole limpet hemocyanin or egg albumin), as described hereafter.

More preferably, the polypeptide or peptide of the present inventionfunctions to promote the formation of a complex of proteins involved inthe fusion of a synaptic vesicle with a presynaptic membrane, such as,for example, the formation of a complex comprising syntaxin-1a, SNAP-25and synaptotagmin. The polypeptide or peptide having such a functionmakes it possible to regulate exocytosis that occurs through the fusionof synaptic vesicles with the presynaptic membrane, by its bindingfunction with human syntaxin-1a. More specifically, an exocytosisregulator containing the polypeptide or peptide is included within thescope of the present invention.

The polypeptide or peptide of the present invention may be a product ina cell in which it is expressed by a genetic engineering technique, asynthesized product of a cell-free system, a chemically synthesizedproduct, or a product prepared from the cell or any biological samples,as well as the further purified products from the foregoing. Further,the polypeptide or peptide of the present invention can be a labeled oneto which another protein or peptide or the like is conjugated at theN-terminal side or C-terminal side thereof, directly or indirectly via alinker peptide or the like by use of a genetic engineering technique, aslong as a function (e.g., binding to syntaxin-1a) of the polypeptide orpeptide of the present invention is not inhibited. Preferably, thelabeling is conducted in a manner that does not inhibit the fundamentalproperties of the polypeptide or peptide. Examples of the protein orpeptide or the like that may be conjugated include an enzyme such asglutathione S-transferase, β-galactosidase, horseradish peroxidase (HRP)or an alkalin phosphatase; tag peptides such as His-tag, Myc-tag,HA-tag, FLAG-tag, or Xpress-tag; a fluorescent substance such as greenfluorescent protein, fluorescein isothiocyanate or phycoerythrin; amaltose binding protein; Fc fragment of immunoglobulin; biotin; and aradioisotope. However, the examples are not limited thereto. Whenmeasuring the protein or peptide or the like itself used in theaforementioned labeling or a function thereof, it is possible to detect,for example, binding of the polypeptide of the present invention tosyntaxin-1a, which makes it easier to detect or purify of thepolypeptide of the present invention.

(Polynucleotide)

According to one embodiment of the present invention, there is provideda polynucleotide containing the nucleotide sequence encoding theaforementioned polypeptide or peptide, or the complementary sequencethereof. For example, the polynucleotide may be a polynucleotidecomprising the nucleotide sequence encoding the polypeptide comprisingthe amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing orthe complementary sequence thereof. Herein, a polynucleotide comprisingthe complementary sequence of a certain polynucleotide is also referredto as a “complementary strand”. These polynucleotides provide, forexample, genetic information that is useful for producing thepolypeptide or peptide described above, or can be used as reagents orreference standards in relation to a nucleic acid.

According to another embodiment of the present invention, there isprovided a polynucleotide that hybridizes under stringent conditions toa corresponding region of a polynucleotide comprising the nucleotidesequence encoding the amino acid sequence of the aforementionedpolypeptide or peptide, for example, the amino acid sequence shown inSEQ ID NO: 1 in the Sequence Listing, or the complementary sequencethereof. The hybridization conditions can be employed in accordancewith, for example, the method shown in the publication (non-patentdocument 5) or the like. These polynucleotides can be those thathybridize to the objective polynucleotide, and need not have acomplementary sequence. For example, these polynucleotides may bepolynucleotides that have homology of preferably 85% or more to thenucleotide sequence of the KIAA1006 gene that encodes the KIAA1006polypeptide, or the complementary sequence thereof, more preferably, ahomology of 90% or more, and further preferably, a homology of 95% ormore.

The polynucleotide of the present invention includes a polynucleotide oroligonucleotide comprising 10 or more consecutive nucleotides located inthe region of the predetermined nucleotide sequence of theabove-mentioned polynucleotide, preferably comprising 15 or moreconsecutive nucleotides, and more preferably comprising 20 or moreconsecutive nucleotides.

The polynucleotide of the present invention may be a polynucleotide towhich a gene (including a gene encoding an enzyme such as glutathioneS-transferase, β-galactosidase, horseradish peroxidase (HRP) or alkalinphosphatase; a tag-peptide such as His-tag, Myc-tag, HA-tag, FLAG-tag orXpress-tag, and the green fluorescent protein) is ligated at the5′-terminal side or 3′-terminal side thereof, as long as, for example,the expression of a polypeptide encoded by the polynucleotide or afunction of the expressed polypeptide is not inhibited thereby.

These polynucleotides are useful for the production of the polypeptideor peptide or the like of the present invention. Further, thesepolynucleotides are also useful as probes or primers for detecting agene encoding the polypeptide of the present invention or mRNA thereof,or as antisense oligonucleotides or the like for regulating theexpression of the gene. In this respect, the polynucleotide of thepresent invention includes not only a translated region but also aregion corresponding to a non-translated region. It is also preferableto use a nucleotide sequence of a unique region in the gene encoding thepolypeptide of the present invention in order to specifically inhibitthe expression of the gene by an antisense oligonucleotide.

Selection of a polynucleotide encoding the polypeptide or peptide of thepresent invention can be carried out, for example, by confirming theexpression of the protein expressed by a known protein-expressionsystem, using in particular, binding to human syntaxin-l a as an indexof a function thereof. For the protein-expression system, for example, acell-free protein-expression system such as a ribosome system derivedfrom wheat germ or rabbit reticulocyte or the like can be used(non-patent document 9).

(Recombinant Vector)

A recombinant vector can be obtained by integrating the polynucleotidedescribed above into an suitable vector DNA. The vector DNA to be usedmay be selected appropriately according to the type of host and thepurpose of use. The vector DNA may be a vector extracted from a naturalsource, or may be a vector that lacks a part of the DNA other than thatnecessary for replication. Examples of the vector DNA include a vectorderived from a chromosome, episome and virus (such as a vector derivedfrom a bacteria plasmid, a bacteriophage, a transposon, a yeast episome,an inserted element, and a yeast chromosome element, for example, aswell as a vector derived from a virus such as baculovirus, papovavirus,SV40, vaccinia virus, adenovirus, fowlpox virus, pseudo-rabies virus andretrovirus), as well as a vector combining the same, such as, forexample, a vector derived from a genetic element of a plasmid andbacteriophage such as a cosmid or a phagemid. In addition, expressionvectors, cloning vectors, and the like can be used depending on thepurpose.

A recombinant vector is composed of the target gene sequence and a genesequence that carries information relating to replication and regulation(such as a promoter, a ribosome binding region, a terminator, a signalsequence and an enhancer and the like), and is produced by combiningthem by using a known method. A method for integrating a polynucleotideinto the vector DNA can be carried out with a known method. For example,a method may be used that comprises: selecting a suitable restrictionenzyme to treat the DNA to cleave it at a specific site, mixing thecleaved DNA with a DNA to be used as a vector that has been treated inthe same manner, and finally re-ligating them with a ligase.Alternatively, a desired recombinant vector can also be obtained byligating a suitable linker to the polynucleotide, and integrated it intothe multiple cloning site of a vector suitable for each purpose.

In the examples described hereinafter, the plasmid hk10084 containingKIAA1006, which was purchased from the Kazusa DNA Research Institute,was used as a template to amplify by PCR the full-length ORF of KIAA1006and a C-terminal truncated product thereof, which were integrated intopFastBacI, respectively, to obtain the baculovirus expression plasmidspFB-1006 (full length) and pFB-1006 (C-terminal truncated product).These two clones were transpositioned to DH10BAC, respectively, toobtain the bacmids Bac-1006 and Bac-1006T. Although a baculovirus vectoris used in the present example, a vector for use in the presentinvention is not limited thereto.

(Transformant)

A transformant can be obtained by transfecting a vector DNA, into whichthe above polynucleotide is integrated, into a known host by a knownmethod. The vector DNA to be transfected into a host may be one kind ofvector DNA or a combination of two or more kinds of vector DNA. Examplesof a host include Escherichia coli, yeast, Bacillus subtilis, insectcells, or animal cells. Introduction of the vector DNA into a host cellmay be carried out by application of a known method, for example, by astandard method shown in the publication (non-patent document 5). Aspreferable systems, the method of integration into chromosomes can becited in consideration of gene stability; however as a simple method, anautonomous replication system using an extranuclear gene can be used.Specifically, examples of the method include calciumphosphate-transfection, DEAE-dextran mediated transfection,microinjection, cationic lipid-mediated transfection, electroporation,genetic transduction, scrape loading, ballistic transfection, andinfection. In the example described hereinafter, an insect cell is used;however a method is not limited thereto (patent 1 and patent 2).

The polypeptides or peptides of the present invention can be providedwhen an expression vector is used as the vector DNA for use intransformation of the host. The transformant (into which an expressionvector DNA containing the aforementioned polynucleotide has beenintegrated) can be cultured under culture conditions and by culturemethods that are suitable for each host and well-known to one skilled inthe art. Culturing can be performed by referring to the function (e.g.,binding to syntaxin 1a) of the polypeptide or peptide of the presentinvention that is expressed by the transformant. Alternatively,culturing can be performed by referring to the quantity of polypeptidesor peptides generated inside or outside the host; passage culture orbatch culturing can also be performed by referring to the quantity oftransformant in the culture medium.

(Collection of the Polypeptide or Peptide)

Collection and/or purification of the polypeptide or peptide of thepresent invention from a culture media in which the transformant wascultured or from the transformant can be carried out by referring to thepresence or absence of a function (e.g., binding to human syntaxin-1a)of the polypeptide or peptide. Examples of a method for recovery and/orpurification include a fractionation method based on differences insolubility using ammonium sulfate or alcohol and the like, gelfiltration, ion column chromatography, and affinity chromatography,which may be used alone or in combination. Preferably, a method can beused wherein polypeptides or peptides are specifically adsorbed andcollected by using polyclonal antibodies or monoclonal antibodies thatcan be prepared against the polypeptides or the peptides based on theinformation of their amino acid sequences.

(Antibody)

An antibody is prepared by using the polypeptide or peptide of thepresent invention as an antigen. An antigen may be the polypeptide orpeptide, or a fragment thereof. The antigen can be composed of at least8, or preferably at least 10, or more preferably at least 12, or furtherpreferably 15 or more amino acids. In order to prepare an antibody thatis specific to the polypeptide and/or peptide, it is preferable to use aregion consisting of the amino acid sequence that is unique to thepolypeptide or peptide. The amino acid sequence of this region does notnecessarily need to be homologous or identical to that of thepolypeptides or peptides. The amino acid sequences of the sites that areexposed to the exterior of the tertiary structure of the polypeptide orpeptide are preferred. Even if the amino acid sequences at the exposedsites are discontinuous in the primary structure, it suffices as far asthey are amino acid sequences that are continuous on the exposed site.The antibody is not particularly limited as long as it is an antibodythat immunologically binds to or recognizes the polypeptide and/orpeptide. The presence or absence of the binding or recognition can bedetermined by a known antigen-antibody binding reaction.

A known antibody production method can be used to produce the antibody.The antibody can be obtained, for example, administering the antigen toan animal with or without linking such to a carrier, in the presence orabsence of an adjuvant, to induce immunity such as a humoral responseand/or cell-mediated response. The carrier is not limited, as long as itdoes not exert any detrimental effects by itself on the host and iscapable of enhancing the antigenicity. Examples of a carrier includecellulose, polymerized amino acids, albumin, and keyhole limpethemocyanin. Examples of an adjuvant that can be used herein includeFreund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA),Ribi (MPL), Ribi (TDM), Ribi (MPL+TDM), whooping cough vaccine(Bordetella pertussis vaccine), muramyl dipeptide (MDP), aluminumadjuvant (ALUM), and combinations thereof. Mice, rats, rabbits, goats orhorses are suitable animals for immunization.

A polyclonal antibody can be obtained from the serum of the animal thatwas treated with the immunization means, by any suitable method forcollecting an antibody. For a preferable method for collecting anantibody, immuno-affinity chromatography method may be exemplified.

A monoclonal antibody can be produced by collecting antibody-producingcells (e.g., lymphocytes derived from spleen or lymph nodes) from ananimal that was treated with the immunizing means, and then carrying outa transformation technique using a permanently proliferating cell (forexample, a myeloma strain such as P3X63Ag8 cells.) For example, theantibody producing cell is fused with a permanently proliferating cellby a known method to form a hybridoma, and then cloning of the hybridomais performed, followed by selecting the hybridoma that produces anantibody recognizing specifically the aforementioned polypeptides and/orpeptides. The antibody can be collected from the cultured solution ofthe hybridoma.

The thus-obtained polyclonal antibody or monoclonal antibody that canrecognize and bind to the polypeptide or peptide of the presentinvention can be used as an antibody for purification of the polypeptideor peptide, a reagent, a labeling marker or the like. Specifically, anantibody that inhibits the function of the polypeptide of the presentinvention can be used for regulating the function of the polypeptide,and is useful for elucidating, preventing, improving and/or treatingmany kinds of disease caused by an abnormality of the polypeptide. Forexample, an antibody that inhibits the binding of the polypeptide tohuman syntaxin-1a can be used, for example, for regulating the releaseof neurotransmitters, and it is therefore useful for elucidating,preventing, improving and/or treating many kinds of disease caused bythe release of an abnormal amount of neurotransmitters.

(Identification and Screening of Compound)

The polypeptide or peptide of the present invention, the polynucleotideof the present invention, the vector into which the polynucleotide isintegrated, the transformant transfected with the vector, the proteinsynthesis system using these, or the antibody that recognizesimmunologically the polypeptide and/or peptide provide means that areuseful for identifying or screening a binding inhibitor or bindingstabilizer that inhibits or stabilizes the binding of the polypeptide orpeptide of the present invention to a protein that interacts with thepolypeptide or peptide, or an expression inhibitor or expressionpromoter that inhibits or promotes the expression of the polynucleotide.The method can be carried out by use of known pharmaceutical screeningsystems. For example, the aforementioned means and method enablescreening for antagonists by means of drug design based on the tertiarystructure of the polypeptide or peptide, screening for an inhibitingagent or a promoting agent of the expression at the gene level using aprotein synthesis system, or screening for a substance recognized by anantibody using the antibody, and the like.

More specifically, it is possible to identify a compound that inhibitsor stabilizes the binding of the polypeptide or peptide of the presentinvention to a protein that interacts with the polypeptide or peptide,by introducing a system to detect the binding of the polypeptide orpeptide of the present invention to a protein that interacts with thepolypeptide or peptide as a signal (marker), and then detecting thepresence, absence or change of the signal (marker) under conditions inwhich the test substance is present or absent.

For example, by referring to the binding of the polypeptide or peptideof the present invention to human syntaxin-1a, a compound can beselected that is capable of influencing the binding. The binding of thepolypeptide or peptide according to the present invention to humansyntaxin-1a can be detected by a binding assay system that uses a knownimmunological method or the like.

For example, a method used in the example described hereunder can beused. In short, the binding of the polypeptide or peptide of the presentinvention to syntaxin-1a can be determined quantitatively by usingGST-fused syntaxin-1a obtained by use of a genetic engineeringtechnique, binding it to glutathione-Sepharose, and determining thebinding of the polypeptide or peptide of the present invention theretousing an antibody against the polypeptide or peptide according to thepresent invention, for example, an antibody labeled with HRP (horseradish peroxidase), alkalin phosphatase, a radioisotope, a fluorescentsubstance or biotin or the like. Alternatively, when using a polypeptideor peptide of the present invention to which a tag-peptide is fused, thequantitative determination can be carried out by use of theanti-tag-antibody. A polypeptide of the present invention that isdirectly labeled with the above-described enzyme, radioisotope,fluorescent substance, biotin or the like may also be used. A secondaryantibody labeled with the above-described enzyme, radioisotope,fluorescent substance, biotin or the like may also be used.

The effect of the test substance on the binding of the polypeptide orpeptide of the present invention to syntaxin-1a can be determined bycomparing the amount of the polypeptide or peptide of the presentinvention bound to syntaxin-1a in the presence of the test substancewith that in the absence of the test substance. When the amount of thepolypeptide or peptide of the present invention that binds tosyntaxin-1a in the presence of the test substance is less than that inthe absence of the test substance, it can be determined that the testsubstance has an effect to inhibit the binding of the polypeptide orpeptide to syntaxin-1a. In contrast, when the amount of the polypeptideor peptide of the present invention that binds to syntaxin-1a in thepresence of the test substance is greater than that in the absence ofthe test substance, it can be determined that the test substance has anactivity to stabilize the binding of the polypeptide or peptide tosyntaxin-1a.

Alternatively, a two-hybrid method that is well known in the art can beused. For example, identification of a test substance can be achieved bytransfecting into yeast or a eukaryotic cell or the like of a plasmidthat expresses the fusion protein of the polypeptide or peptide of thepresent invention and a DNA-binding protein, a plasmid that expressesthe fusion protein of syntaxin-1a and a transcription activationprotein, and a plasmid containing a reporter gene such as lacZ with aconnected suitable promoter gene, and then comparing the expressionamount of the reporter gene in the presence of the test substance withthat in the absence of the test substance. When the expression amount ofthe reporter gene in the presence of the test substance is less thanthat in the absence of the test substance, it can be determined that thetest substance has an activity to inhibit the binding of the polypeptideor peptide to syntaxin-1a. In contrast, when the expression amount ofthe reporter gene in the presence of the test substance is greater thanthat in the absence of the test substance, it can be determined that thetest substance has an activity to stabilize the binding of thepolypeptide or peptide to syntaxin-1a.

It is possible to use a surface plasmon resonance sensor, such asBIACORE system, to identify a compound that influences the binding ofthe polypeptide or peptide of the present invention to syntaxin-1a.

It is also possible to use a method in which a scintillation proximityassay (SPA) or a fluorescence resonance energy transfer (FRET) isapplied, to identify a compound that influences the binding of thepolypeptide or peptide of the present invention to syntaxin-1a.

The syntaxin-1a used in these screening systems can be a protein inwhich a part thereof has been deleted or to which another protein hasbeen added, as long as it does not influence the binding to thepolypeptide or peptide of the present invention.

It is possible to identify a compound that inhibits or stabilizes theexpression of the polynucleotide of the present invention, by selectingconditions that allows the expression of the polynucleotide, contactingthe polynucleotide with a test compound under those conditions,introducing a system that uses a signal capable of detecting thepresence or the absence of the expression, and detecting the presence orabsence or change of the signal. As a signal to be used herein,tag-proteins such as glutathione S-transferase, His-tag, Myc-tag,HA-tag, FLAG-tag or Xpress-tag and green fluorescent protein or the likeare available.

Alternatively, a compound that influences the expression of a gene ofthe present invention can be identified by, for example, preparing avector in which a reporter gene, in place of the gene encoding thepolypeptide of the present invention, is connected downstream of thepromoter region of the gene e, then contacting the test substance with acell (such as an eukaryotic cell) into which the vector is transfected,and finally detecting the presence, absence or change of expression ofthe reporter gene. A gene that is generally used in a reporter assay canbe used as the reporter gene; for example, a gene having enzyme activitysuch as luciferase, β-galactosidase or chloramphenicol acetyltransferase can be used. Detection of the expression of the reportergene can be carried out by detecting the activity of the gene product.In the case of using the gene exemplified above, detection of theexpression of the reporter gene can be carried out by detecting theenzyme activity.

(Compound)

A compound identified as described above is available as a candidatecompound for a binding inhibitor, binding antagonist, or bindingstabilizer relating to a polypeptide of the present invention. Thosecompounds are also available as candidate compounds for an expressioninhibitor or expression stimulator for the aforementioned polypeptide ata gene level. It can be expected that these candidate compounds willhave an effect of preventing and/or treating many kinds of diseasescaused by an increase, decrease or lack of expression or activity of thepolypeptide of the present invention. For example, a candidate compoundcan be used for regulating the release of a neurotransmitter, so that itcan be expected to have an effect of preventing and/or treating diseasescaused by abnormal release of neurotransmitters and an abnormal amountof released neurotransmitters.

(Pharmaceutical Composition)

The thus-selected candidate compounds can be used to preparepharmaceutical compositions by further selecting, with considerationgiven to the balance between the biological effectiveness and toxicitythereof. The polypeptide or peptide of the present invention, thepolynucleotide of the present invention, the plasmid of the presentinvention, and the antibody that immunologically recognizes thepolypeptide or peptide can be used themselves as a diagnosis means (suchas a diagnosis marker or reagent), as well as a pharmaceutical meanssuch as therapeutics (e.g., an inhibitor, an antagonist, or stimulatorthat utilizes the function of inhibiting, antagonizing, or promoting theactivity and/or expression of the polypeptide according to the presentinvention). More specifically, the present invention provides apharmaceutical composition containing at least one of the abovesubstances, by utilizing one of them or by utilizing a combination ofthem.

When the expression of the polypeptide of the present invention and theactivity thereof are excessive, an effective amount of theaforementioned inhibitor can be administrated with a pharmaceuticallyacceptable carrier to inhibit the activity of the polypeptide andthereby improve the abnormal symptoms. Further, it is possible toinhibit the expression of the endogenous gene that encodes thepolypeptide by using an expression blocking method. The expression ofthe gene can be inhibited by using an antisense sequence of the genethat is made to be produced in a cell or is particularly administrated.These oligonucleotides can be designed and synthesized on the basis ofthe polynucleotide of the present invention. The oligonucleotide itselfcan be administrated, or related oligomers can be expressed in vivo.

For treatment of an abnormal symptom relating to a decrease or lack ofexpression of the polypeptide of the present invention and activitythereof, an effective amount of the agent for promoting the activitiesof the gene encoding the polypeptide or peptide can be administratedwith a pharmaceutically acceptable carrier and thereby improve theabnormal symptom. Alternatively, it is possible to produce thepolypeptide in the cell of the target by use of gene therapy. Genetherapy using the polynucleotide of the present invention can beperformed according to a known method. For example, areplication-deficient retrovirus vector into which the polynucleotide ofthe present invention has been integrated may be prepared and used ingene therapy. It is also possible in gene therapy to produce a proteinused in the therapy in the target itself. For example, using DNA or RNAthat encodes the protein, a cell derived from the target can be treatedex vivo by use of, e.g., a retrovirus plasmid vector, and then the cellcan then be introduced into the target.

The polypeptide of the present invention binds to syntaxin-1a, and thus,it is considered that in particular, the polypeptide relates to adisease caused by an abnormal release of neurotransmitters or anabnormal amount thereof. Therefore, the inhibitor or antagonistmentioned above is useful for preventing and/or treating the disease. Amethod for promoting or inhibiting the release of neurotransmitters,wherein the method comprises inhibiting or stabilizing binding of apolypeptide of the present invention to syntaxin-1a, and a method forpreventing and/or treating a disease caused by an abnormal amount ofneurotransmitters are included within the scope of the presentinvention.

In terms of a formulation, the pharmaceutical composition of the presentinvention is preferably formulated in combination with a suitablepharmaceutical carrier. Such a formulation comprises a therapeuticallyeffective amount of at least one member selected from the groupconsisting of the polypeptide or peptide, the polynucleotide, thevector, the transformant, the antibody and the compound of the presentinvention, and a pharmaceutically acceptable carrier or vehicle.Examples of a carrier include physiological saline, bufferedphysiological saline, dextrose, water, glycerol, ethanol, and a mixturethereof; however, it is not limited thereto. The formulation may beselected according to an administration route, and such formulations arewell-known to those skilled in the art. At the time of formulation, thecompositions may be used alone or in combination with other compounds ormedicine required for treatment.

In terms of the mode of administration, it may be either systemicadministration or local administration. One preferred mode of systemicadministration is injection, e.g., an intravenous injection. Otherinjection routes, such as subcutaneous, intramuscular, orintraperitoneal injection, may also be used. Another mode ofadministration may be oral administration, so long as an entericformulation or a capsule formulation can be suitably formulated. Inaddition, transmucosal administration or percutaneous administration,which comprises using a penetrant such as bile salt, fusidic acid, orother surfactants, may also be used. In a local administration, formssuch as ointment, paste, or gel, may be used.

Suitable dosage ranges can be determined according to the following:effectiveness of a therapeutically effective amount of at least onemember selected from the group consisting of the polypeptide or peptide,the polynucleotide, the vector, the transformant, the antibody and thecompound of the present invention; the route of administration; theproperties of the formulation; the symptomatic conditions of thesubject; and the judgment of the doctor in charge. More specifically, asuitable dosage may fall within the range of, e.g., 0.1 μg to 10 μg per1 kg of the body weight of the subject. However, the dosage may bealtered using common conventional experiments for optimization of adosage that are well known in the art.

Preparation of a pharmaceutical may be carried out by well-knownformulating procedures in accordance with the physical properties of thesubstance employed, for example, peptides, proteins, oligonucleotides,antibodies, or compounds. Specifically, formulations such as powdereddrugs, pills, tablets, capsules, aqueous solutions, liposomalformulations, fat emulsions, clathrates (such as those of cyclodextrin),and the like can be used.

Powdered drugs, pills, capsules, or tablets can be prepared using, forexample, an excipient such as lactose, glucose, sucrose, or mannitol; adisintegrant such as starch or sodium arginate; a lubricant such asmagnesium stearate or talc; a binder such as polyvinylalcohol,hydroxypropyl cellulose, or gelatin; a surfactant such as fatty acidester; and a plasticizer such as glycerin, or the like. For preparationof a tablet or capsule, a solid pharmaceutical carrier is used.

A suspension can be prepared using water; saccharides such as sucrose,sorbitol, or fructose; glycols such as PEG; and oils.

Injectable solutions can be prepared using a carrier comprising a salinesolution, a glucose solution, or a mixture of the salt water and glucosesolution.

Inclusion into a liposome formulation may be conducted in the followingmanner: by dissolving the substance of interest in a solvent (e.g.,ethanol) to make a solution, adding a solution of phospholipidsdissolved in an organic solvent (e.g., chloroform), removing the solventby evaporation and adding a phosphate buffer thereto, agitating thesolution and then subjecting it to sonication followed by centrifugationto obtain supernatant, and finally, filtrating the supernatant forrecovering a liposome.

Fat emulsion can be prepared in the following manner: by mixing thesubstance of interest, an oil ingredient (vegetable oil such as soybeanoil, sesame oil, olive oil, or MCT), an emulsifier (such asphospholipid), and the like; heating the mixture to make a solution;adding water of a required quantity; and then emulsifying orhomogenizing by use of an emulsifier (homogenizer, e.g., a high pressurejet type, an ultrasonic type, or the like). The fat emulsion may be alsolyophilized. For conducting lipid-emulsification, an auxiliaryemulsifier may be added, and examples thereof include glycerin orsaccharides (e.g., glucose, sorbitol, fructose, etc.).

Inclusion into a cyclodextrin formulation may be carried out in thefollowing manner: by dissolving the substance of interest in a solvent(e.g., ethanol); adding a solution of cyclodextrin dissolved in waterunder heating thereto; chilling the solution and filtering theprecipitates; and drying under sterilization. At this time, thecyclodextrin to be used may be appropriately selected from among thosehaving different void sizes (α, β, or γ type) in accordance with thebulkiness of the substance.

(Measurement Method and Reagent for Diagnosis)

The polypeptide or peptide of the present invention, polynucleotide ofthe present invention, or the antibody that immunologically recognizesthe polypeptide or peptide can be used itself as a marker for diagnosisor a reagent. When used as reagents, they may contain a substance suchas a buffer, salt, stabilizer, and/or antiseptic agent. The presentinvention also provides a reagent kit comprising one or more containersin which one or more of the substances mentioned above are filled. Interms of the preparation thereof, it is sufficient to use a well-knownmeans for the preparation suitable for a polypeptide or peptide,protein, polynucleotide, or an antibody, respectively.

These reagents and reagent kits can be used in the identification methodof the present invention. The reagents and reagent kits can also be usedfor quantitative and/or qualitative measurement of the polypeptide orpeptide of the present invention, or a polynucleotide encoding eitherone thereof. A method for performing the measurement can be constructedusing a method that is known to those skilled in the art. Examples ofmethods that can be used in the measurement of the polypeptide orpeptide include radioimmunoassay, competitive binding assay, Westernblotting assay and ELISA assay. The polynucleotide can be detected andquantitatively determined at the nucleic acid level, for example, theRNA level, by use of, for example, amplification, PCR, RT-PCR, RNaseprotection, Northern blotting, and other methods of hybridization.

Further, the test or diagnosis of a disease caused by the expression oractivity of the polypeptide is enabled by qualitatively orquantitatively measuring the polypeptide or peptide of the presentinvention or a nucleic acid encoding one of these as a diagnosis marker.A method for the test or the diagnosis can be performed, for example, byusing the interaction or reactivity with the polynucleotide encoding theaforementioned polypeptide or peptide to detect the presence of thecorresponding nucleic acid, to determine the existing amount thereof,and/or to identify a mutation thereof, as well as by determining the invivo distribution of the polypeptide or peptide in an individual, bydetecting the presence of the polypeptide or peptide, by determining theexisting amount thereof, and/or by detecting a mutation thereof.

Examples of a sample include a sample derived from an individual such asa cell, blood, urine, saliva, spinal fluid, tissue biopsy or autopsyspecimen. A nucleic acid can be obtained from each sample mentionedabove by a known method. A nucleic acid can be used directly fordetection in the form of genome DNA, or may be used after beingenzymatically amplified by PCR or other amplifying methods before theanalysis. RNA or cDNA may also be used in the same way. A deletion orinsertion can be detected by a change in size of an amplified productwhen compared to the size of the normal gene type. A point mutation canbe identified by hybridization of the amplified DNA to a labeled DNAencoding the polypeptide of the present invention.

Detecting a mutation, decrease or increase in the polypeptide of thepresent invention and a nucleic acid encoding the polypeptide, enablesdiagnosis of a disease caused by the abnormal release ofneurotransmitters or an abnormal amount thereof.

EXAMPLES

Hereinafter, the present invention may be explained more particularlywith an example; however, the present invention is not limited to thefollowing example.

(Isolation of Genes)

A homology search was performed for a clone having homology to the VAMPhomologous region of rat m-tomosyn using the human brain-derived longchain cDNA library database of the Kazusa DNA Research Institute. As aresult, it was found that KIAA1006 has homology to rat m-tomosyn ofabout 57.6% at the nucleotide level and of about 67.9% at the amino acidlevel.

In the database of the Kazusa DNA Research Institute, it was pointed outthat KIAA1006 may be a partial sequence with a N-terminal truncation.Therefore, in order to determine the presence of a translationinitiation site in the upstream region of KIAA1006 gene, 5′-rapidamplification of cDNA ends (RACE) was carried out using human polyA(+)RNA (Clontech Laboratories, Inc.), 1006 race primer (SEQ ID NO: 2 in theSequence Listing) and SMART RACE cDNA Amplification Kit (ClontechLaboratories, Inc.). 1006 race: GCCAGGGGAGGAGGCAGTTAAGCCATC (SEQ ID NO:2 in the Sequence Listing).

As a result, no novel upstream sequences were found. Further, inconsideration of the fact that the amino acid sequence following thefirst methionine within the amino acid sequence deduced from thenucleotide sequence of KIAA1006 showed a remarkably high homology to theN-terminal sequence of rat m-tomosyn at amino acid 1-15, it wasdetermined that the first methionine of KIAA1006 is the translationinitiation site of the gene containing KIAA1006, and the followinganalysis was done.

(Construction of expression plasmids for full-length ORF of KIAA1006 andC-terminal-truncated ORF of KIAA1006).

Plasmid hk10084 containing KIAA1006 was purchased from the Kazusa DNAResearch Institute. Plasmid hk10084 is a plasmid produced by insertingKIAA1006 into Sal I-Not I site of pBluescript II SK(+) vector.Specifically, Plasmid hk10084 is a plasmid produced by ligating Sal Iadaptor to the 5′ terminal of KIAA1006 cDNA to insert the ligated DNAinto the Sal I site of pBluescript II SK(+) vector, and inserting the 3′terminal of KIAA1006 cDNA into the Not I site of the vector. Plasmidhk10084 was used as a template. The ORF region of KIAA1006 was amplifiedusing the combination of 1006 (s) (SEQ ID NO: 3 in the Sequence Listing,including the His×6-Tag sequence) and 1006 (a)1 (SEQ ID NO: 4 in theSequence Listing, for the full length ORF), or the combination of 1006(s) (SEQ ID NO: 3 in the Sequence Listing) and 1006 (a)2 (SEQ ID NO: 5in the Sequence Listing) (for the C-terminal truncated product), andAdvantage-HF2 PCR Kit (Clontech Laboratories, Inc.). The two PCRproducts thus obtained were digested with EcoRI and XhoI and insertedinto pFastBacI (Gibco BRL Inc.) that had been treated with the sameenzymes, to obtain baculovirus expression plasmids, pFB-1006 (encodingfull length amino acid 1-1186) and pFB-1006t (encoding amino acid1-1157, in which the inferred VAMP homology region was deleted from thefull length amino acid). The respective bacmids, Bac-1006 and Bac-1006t,were obtained by transposition of these plasmids into competent E. ColiDH10BAC (Gibco BRL Inc.).

1006(s):CCGGAATTCGCCACCATGCATCACCATCACCATCACAAGAAGAAGTTTA ATTTCCGAAAAGTT(SEQ ID NO: 3 in the Sequence Listing)

1006(a)1:CCGCTCGAGTCAGAATTGGTACCATTTCTTATCCTTG (SEQ ID NO: 4 in theSequence Listing)

1006(a)2:CCGCTCGAGTCAAGTTTTCTCTTCCAGCTCTCCTAG (SEQ ID NO: 5 in theSequence Listing)

As used herein, “(s)” denotes a sense primer and “(a)” denotes anantisense primer.

(Expression and Purification of KIAA1006 Polypeptide and KIAA1006C-terminal Truncated Polypeptide in sf9 Cell).

Bac-1006 and Bac-1006t were transfected into sf9 cells (35 mm dish),respectively, by use of CellFECTIN (Gibco BRL Inc.). The culturesupernatant after 3 days from the transfection was used for re-infectionof the sf9 cells to amplify the baculovirus. sf9 cells were subjected toinfection with the amplified virus, and 3 days later, the cells weredissolved in EM buffer (50 mM sodium phosphate/300 mM NaCl/1% NonidetP-40/1 mM phenylmethanesulfonyl fluoride (PMSF)), and subjected toultrasonic disintegration. Histidine-tagged KIAA1006 polypeptide andhistidine-tagged KIAA1006 C-terminal truncated polypeptide were purifiedfrom the extraction of the obtained disintegrated cell using a cobaltresin (TALON, Clontech Laboratories, Inc.). By Western blotting using ananti-His antibody (Qiagen Inc.), it was confirmed that thehistidine-tagged KIAA1006 polypeptide and histidine-tagged KIAA1006C-terminal truncated polypeptide were recovered in almost the sameamount.

It has been reported that rat m-tomosyn was expressed in sf9 cells andCOS7 cells because rat m-tomosyn could not be expressed by a commonmethod using E. coli (non-patent document 1). However, it has been alsoreported that even by this method, the protein obtained was insoluble,so that a protein that was renatured after being subjected to SDS-PAGEwas used for analysis. The two forms of the polypeptide of the presentinvention that were found by the inventors could be purified as solubleproteins and could be purified using a cobalt resin, which made itpossible to conduct an in-vitro binding test. The application of thepresent methods of expression and purification makes it possible tomeasure the binding/dissociation constant or the like between humansyntaxin-1a and a polypeptide of the present invention, which wasimpossible for rat m-tomosyn by Fujita et al. (non-patent document 1).

(Expression and Purification of Human Syntaxin-1a in E. coli)

To obtain human syntaxin-1a employing cDNA derived from human brainpolyA RNA (Clontech Laboratories, Inc.) as a template, 1-266AA withinthe ORF region of syntaxin-1a was amplified by PCR using the twoprimers, sy1a4T(s) (SEQ ID NO: 6 in the Sequence Listing) and sy1a4T(a)(SEQ ID NO: 7 in the Sequence Listing) described below. The amplifiedproducts were digested with EcoRI and XhoI, and the digested productswere then integrated into a plasmid for expression of the GST-fusionprotein, pGEX-4T-3 (Amersham Pharmacia Biotech), that had been digestedwith EcoRI and XhoI in a similar manner, to obtain pGEX-sy1a. Thesequence of pGEX-sy1a was determined, and the absence of mutagenesis orframe-shift caused by PCR was confirmed.

sy1a4T(s):CCGGAATTCCATGAAGGACCGAACCCAGGAGCTC (SEQ ID NO: 6 in theSequence Listing)

sy1a4T(a):CCGCTCGAGTTACGCCTTGCTCTGGTACTT (SEQ ID NO: 7 in the SequenceListing)

DH5a was transformed with pGEX-sy1a and cultured at 37° C. until itentered the late proliferation phase (OD 0.5 or more). Isopropylβ-D-thiogalactopyranoside (IPTG) was added thereto at the finalconcentration of 1 mM, and 5 hours of expression induction was carriedout. The collected bacteria was washed with PBS(-), suspended in BufferA (50 mM tris HCl pH 7.5/1 mM EDTA/1 mM DTT/1 mM PMSF), and then treatedfor 30 min with lysozyme (0.5 mg/ml) in ice water. After adding NonidetP-40 (final concentration of 1%), the bacteria was sonicated, andGST-syntaxin-1a (1-266AA) was purified by the use ofglutathione-Sepharose according to a common method from the supernatantobtained after centrifugation. Expression induction was carried out in asimilar manner for pGEX-4T-3 having no insertion to express and purifyGST.

Syntaxin-1a that was expressed herein was not the full length of theORF, but a protein consisting of 1-266 amino acids corresponding tosyntaxin-1a with C-terminal truncation. Syntaxin-1a with C-terminaltruncation consisting of 1-266 amino acids was used, since it wasconfirmed in the reports (non-patent document 10, non-patent document11, non-patent document 12, and non-patent document 13) that theextremely low hydrophilicity of the 21 amino acid residues in theC-terminal side of syntaxin-1a were involved in a problem ofinsolubilization, and C-terminal truncation did not reduce theproperties of syntaxin-1a as a protein.

As a result, a purified standard product of GST-syntaxin-1a fusedprotein, which became almost a single band by Coomassie brilliant bluestaining, was obtained.

(Analysis of Binding of KIAA1006 Polypeptide with Syntaxin-1a)

KIAA1006 polypeptide (the histidine-tagged KIAA1006 polypeptidedescribed above) and its C-terminal truncated product (thehistidine-tagged C-terminal truncation product of KIAA1006 polypeptidedescribed above) purified with a cobalt resin were respectively mixedwith glutathione-Sepharose (bed volume 10 μl) to which GST (17 μg) wasbound, stirred for 6 hours at 4° C., and then centrifuged. The obtainedsupernatants (GST-glutathione-Sepharose unadsorbed fractions) wererespectively added to glutathione-Sepharose (bed volume 10 μl) to whichGST-syntaxin-1a (17 μg) was bound, and after stirring for 16 hours at 4°C., the resultants were subjected to centrifugation. The fractionsadsorbed to GST-glutathione-Sepharose and the fractions adsorbed toGST-syntaxin-1a-glutathione-Sepharose were eluted with SDS-PAGE samplebuffer, respectively, and the binding of syntaxin-1a to KIAA1006polypeptide or to its C-terminal truncation product was analyzed byWestern blotting using anti-penta His antibody (Qiagen).

For the SDS-PAGE, 4-12% Bis-Tris Gel (NOVEX) was used. The detection ofa secondary antibody that was labeled with horseradish peroxidase wascarried out using ECL Western blotting detection reagents (AmershamPharmacia Biotech).

As a result, as shown in FIG. 2, in the GST-glutathione-Sepharoseadsorbed fractions, neither KIAA1006 polypeptide nor the C-terminaltruncated product was detected (FIG. 2: lanes 1 and 2). In contrast, inthe elutions from glutathione-Sepharose bound with GST-syntaxin-1a , aband that was detectable by anti-penta-His antibody was detected whenKIAA1006 polypeptide was applied, but the C-terminal truncated productwas not detected in the elution when the C-terminal truncated productwas applied (FIG. 2: lanes 4 and 6).

Thus, it was revealed that the full length ORF of KIAA1006 binds tosyntaxin-1a and that the C-terminal truncation product does not bind tosyntaxin-1a. From these results, it was clarified that KIAA1006polypeptide binds to syntaxin through the VAMP homologous regionthereof.

INDUSTRIAL APPLICABILITY

As described above, it was found that the clone KIAA1006, which wasselected from a human brain-derived long chain cDNA library disclosed bythe Kazusa DNA Research Institute, encodes a protein that binds to humansyntaxin-1a. This gene has homology to rat m-tomosyn of about 57.6% atthe nucleotide level and of about 67.9% at the amino acid level. It hasbeen reported on a database that KIAA1006 is specifically expressed inthe brain. It is known that syntaxin-1a is an important protein forreleasing neurotransmitters, which suggest that KIAA1006 polypeptide,which is capable of binding to syntaxin-1a, also plays an important rolein the release of neurotransmitters. Thus, it has been found thatKIAA1006 according to the present invention, a polypeptide derivedtherefrom, and polynucleotides encoding these are useful for apreventive and/or therapeutic medicine for a disease relating to thepolypeptide of the present invention, for example, a disease caused byabnormal release of neurotransmitters or an abnormal amount thereof, aswell as for a diagnosis means.

Sequence Listing Free Text.

SEQ ID NO: 1 in the Sequence Listing: an amino acid sequence ofpolypeptide consisting of full length KIAA1006 ORF, deduced from itscDNA sequence.

SEQ ID NO: 2 in the Sequence Listing: a primer used for analysing theupstream of N-terminus of KIAA1006 by the 5′-race method.

SEQ ID NO: 3 in the Sequence Listing: a sense primer used for amplifyingthe partial sequence of KIAA1006 ORF by PCR.

SEQ ID NO: 4 in the Sequence Listing: an antisense primer used foramplifying the partial sequence of KIAA1006 ORF by PCR.

SEQ ID NO: 5 in the Sequence Listing: an antisense primer used foramplifying the partial sequence of a C-terminal deletion mutant ofKIAA1006 ORF by PCR.

SEQ ID NO: 6 in the Sequence Listing: a sense primer used for amplifyingthe sequence of syntaxin 1a by PCR.

SEQ ID NO: 7 in the Sequence Listing: an antisense primer used foramplifying the sequence of syntaxin-1a by PCR.

1. A polypeptide having a function of binding to human syntaxin-1a,wherein the polypeptide is selected from the group consisting of: (i) apolypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 inthe Sequence Listing; (ii) a polypeptide containing the polypeptide of(i); (iii) a polypeptide having at least 90% homology to the amino acidsequence of the polypeptide of (i), and (iv) a polypeptide having amutation such as a deletion, substitution, addition or insertion of oneor several amino acids relative to the amino acid sequence of any of thepolypeptides of (i) to (iii).
 2. A peptide comprising at least fiveconsecutive amino acid residues in the amino acid sequence shown in SEQID NO: 1 in the Sequence Listing.
 3. The polypeptide according to claim1, wherein the polypeptide promotes the formation of a complex thatcomprises human syntaxin-1a, synaptosomal-associated 25 kDa protein andsynaptotagmin and is involved in fusion of a synaptic vesicle with apresynaptic membrane.
 4. A regulator of exocytosis through fusion of asynaptic vesicle with a presynaptic membrane, wherein the regulatorcomprises the polypeptide according to claim
 1. 5. A polynucleotidecomprising a nucleotide sequence that encodes the polypeptide accordingto claim 1, or the complementary sequences thereof.
 6. A polynucleotidethat hybridizes to the polynucleotide according to claim 5 understringent conditions.
 7. A polynucleotide comprising at least 15consecutive nucleotides of the polynucleotide according to claim
 5. 8. Arecombinant vector containing the polynucleotide according to claim 5 ora polynucleotide that hybridizes to said polynucleotide under stringentconditions or a polynucleotide comprising at least 15 consecutivenucleotides of said polynucleotide.
 9. The recombinant vector accordingto claim 8, wherein the recombinant vector is a recombinant expressionvector.
 10. A transformant that is transformed with the recombinantvector according to claim
 8. 11. A method for producing the polypeptideaccording to claim 1, wherein the method comprises a step of incubatinga transformant transformed with a recombinant expression vectorcontaining a polynucleotide comprising a nucleotide sequence thatencodes the polypeptide.
 12. An antibody that immunologically recognizesthe polypeptide according to claim
 1. 13. An antibody thatimmunologically recognizes the polypeptide according to claim 1 and thatinhibits the binding of said polypeptide to a protein that interactswith said polypeptide.
 14. An antibody that immunologically recognizesthe polypeptide according to claim 1 and that inhibits the binding ofsaid polypeptide to human syntaxin-1a.
 15. A method comprisingidentifying a compound that inhibits or stabilizes the binding of thepolypeptide according to claim 1 or a peptide comprising at least fiveconsecutive amino acid residues of the amino acid sequence of SEQ ID NO:1 to a protein that interacts with said polypeptide or said peptideand/or a compound that inhibits or promotes expression of apolynucleotide comprising a nucleotide sequence that encodes saidpolypeptide, wherein the method uses at least one member selected fromthe group consisting of said polypeptide or a peptide comprising atleast five consecutive amino acid residues of the amino acid sequence ofSEQ ID NO: 1, a polynucleotide comprising a nucleotide sequence thatencodes said polypeptide, a recombinant vector containing apolynucleotide comprising a nucleotide sequence that encodes saidpolypeptide or a polynucleotide that hybridizes to said polynucleotidecomprising a nucleotide sequence that encodes said polypeptide, or apolynucleotide that comprises at least 15 consecutive nucleotides of anucleotide sequence that encodes said polypeptide, a transformanttransformed with a recombinant vector containing one of saidpolynucleotides, and an antibody that immunologically recognizes saidpolypeptide or a peptide comprising at least five consecutive amino acidresidues of said amino acid sequence of SEQ ID NO:
 1. 16. A method foridentifying a compound that inhibits or stabilizes the binding of thepolypeptide according to claim 1 to a protein that interacts with saidpolypeptide, wherein the method comprises steps of: contacting a testsubstance with said polypeptide and the protein that interacts with saidpolypeptide under conditions that allow the binding of said polypeptideto the protein that interacts with said polypeptide; and determiningwhether the test substance inhibits or stabilizes the binding of saidpolypeptide to a protein that interacts with said polypeptide bydetecting the presence, absence, or change of a signal generated by thebinding of said polypeptide to the protein that interacts with saidpolypeptide.
 17. A method for identifying a compound that inhibits orstabilizes the binding of the polypeptide according to claim 1 to humansyntaxin-1a, wherein the method comprises steps of: contacting a testsubstance with said polypeptide and human syntaxin-1a, under conditionsthat allow the binding of said polypeptide to human syntaxin-1a; anddetermining whether the test substance inhibits or stabilizes thebinding of said polypeptide to human syntaxin-1a, by detecting thepresence, absence, or change of a signal generated by the binding ofsaid polypeptide to human syntaxin-1a.
 18. A method for identifying acompound that inhibits or promotes expression of the polynucleotideaccording to claim 5, wherein the method comprises steps of: contactinga test substance with said polynucleotide under conditions that allowexpression of said polynucleotide; and determining whether the testsubstance inhibits or promotes expression of said polynucleotide bydetecting the presence, absence, or change of a signal generated byexpression of said polynucleotide.
 19. A compound that is identified bythe method according to claim
 15. 20. A compound that inhibits orstabilizes the binding of the polypeptide according to claim 1 to humansyntaxin-1a by interacting with said polypeptide.
 21. A compound thatinhibits or promotes expression of the polynucleotide according to claim5 by interacting with said polynucleotide.
 22. A pharmaceuticalcomposition comprising at least one member selected from the groupconsisting of the polypeptide according to claim 1 a peptide comprisingat least five consecutive amino acid residues in the amino acid sequenceof SEQ ID NO: 1, a polynucleotide comprising a nucleotide sequence thatencodes said polypeptide or a polynucleotide that hybridizes understringent conditions to said polynucleotide that comprises a nucleotidesequence that encodes said polypeptide or a polynucleotide comprising atleast 15 consecutive nucleotides of a nucleotide sequence that encodessaid polypeptide or complementary sequences thereof, a recombinantvector containing a polynucleotide comprising a nucleotide sequence thatencodes said polypeptide or a polynucleotide that hybridizes understringent conditions to the polynucleotide that comprises a nucleotidesequence that encodes said polypeptide or a polynucleotide comprising atleast 15 consecutive nucleotides of a nucleotide sequence that encodesfor said polypeptide or complementary sequences thereof, a transformanttransformed with said recombinant vector, an antibody thatimmunologically recognizes said polypeptide, and a compound thatinhibits or stabilizes the binding of said polypeptide or a peptidecomprising at least five consecutive amino acid residues in the aminoacid sequence of SEQ ID NO: 1 to a protein that interacts with saidpolypeptide or peptide and/or a compound that inhibits or promotesexpression of a polynucleotide comprising a nucleotide sequence thatencodes said polypeptide.
 23. The pharmaceutical composition accordingto claim 22 that has a function to promote or inhibit release ofneurotransmitters.
 24. The pharmaceutical composition according to claim22, which prevents and/or is a remedy for a disease caused by anabnormal amount of neurotransmitters.
 25. A method for promoting orinhibiting release of neurotransmitters, wherein the method comprisesinhibiting or stabilizing the binding of the polypeptide according toclaim 1 to human syntaxin-1a.
 26. A method for preventing or treating adisease caused by an abnormal amount of neurotransmitters, wherein themethod comprises inhibiting or stabilizing the binding of thepolypeptide according to claim 1 to human syntaxin-1a.
 27. A methodcomprising quantitatively or qualitatively measuring a polypeptideaccording to claim 1 or a polynucleotide encoding said polypeptide
 28. Amethod for measurement used in diagnosis of a disease caused by anabnormality in the expression or function of the polypeptide accordingto claim 1, wherein the method comprises conducting an analysisemploying (A) a nucleic acid encoding said polypeptide and/or (B) saidpolypeptide as a marker.
 29. A method for measurement used in diagnosisof a disease associated with an abnormal function of neurotransmittioncaused by an abnormal amount of the polypeptide according to claim 1,wherein the method comprises conducting an analysis employing (A) anucleic acid encoding said polypeptide and/or (B) said polypeptide as amarker.
 30. A reagent kit comprising at least one member selected fromthe group consisting of the polypeptide according to claim 1 or apeptide comprising at least five consecutive amino acid residues in theamino acid sequence of SEQ ID NO: 1, a polynucleotide comprising anucleotide sequence that encodes said polypeptide or a polynucleotidethat hybridizes under stringent conditions to said polynucleotide thatcomprises a nucleotide sequence that encodes said polypeptide or apolynucleotide comprising at least 15 consecutive nucleotides of anucleotide sequence that encodes said polypeptide or complementarysequences thereof, and a recombinant vector containing saidpolynucleotide comprising a nucleotide sequence that encodes saidpolypeptide according to claim 1, a transformant transformed with arecombinant vector containing said polynucleotide comprising anucleotide sequence that encodes said polypeptide or a polynucleotidethat hybridizes under stringent conditions to said polynucleotide thatcomprises a nucleotide sequence that encodes said polypeptide or apolynucleotide comprising at least 15 consecutive nucleotides of anucleotide sequence that encodes said polypeptide or complementarysequences thereof, and an antibody that immunologically recognizes saidpolypeptide or a peptide comprising at least five consecutive amino acidresidues in the amino acid sequence of SEQ ID NO:
 1. 31. The reagent kitaccording to claim 30, which is used in a method for identifying acompound that inhibits or stabilizes the binding of the polypeptideaccording to claim 1 to a protein that interacts with said polypeptideand/or a compound that inhibits or promotes expression of apolynucleotide comprising a nucleotide sequence that encodes saidpolypeptide, or in a measurement method for quantitatively orqualitatively measuring said polypeptide or a polynucleotide encodingsaid polypeptide.
 32. A polynucleotide comprising a nucleotide sequenceencoding the polypeptide according to claim 1, excluding a DNAnucleotide sequence registered in GenBank under accession numberAB023223, or the complementary sequence thereof.
 33. An agent forpromoting the formation of a complex that comprises human syntaxin-1a,synaptosomal-associated 25 kDa protein and synaptotagmin andparticipates in fusion of a synaptic vesicle with a presynapticmembrane, wherein the agent comprises the polypeptide according toclaim
 1. 34. An inhibitor comprising an antibody that immunologicallyrecognizes the polypeptide according to claim 1 and that inhibits thebinding of said polypeptide to a protein that interacts with saidpolypeptide.
 35. An inhibitor comprising an antibody thatimmunologically recognizes the polypeptide according to claim 1 and thatinhibits the binding of said polypeptide to human syntaxin-1a.
 36. Thepeptide according to claim 2, wherein the peptide promotes the formationof a complex that comprises human syntaxin-1a, synaptosomal-associated25 kDa protein and synaptotagmin and is involved in fusion of a synapticvesicle with a presynaptic membrane.
 37. A regulator of exocytosisthrough fusion of a synaptic vesicle with a presynaptic membrane,wherein the regulator comprises the peptide according to claim 2 and hasa binding function to human syntaxin-1a.
 38. A polynucleotide comprisinga nucleotide sequence that encodes the peptide according to claim 2, orthe complementary sequences thereof.
 39. A polynucleotide thathybridizes to the polynucleotide according to claim 38 under stringentconditions.
 40. A recombinant vector containing the polynucleotideaccording to claim
 38. 41. A recombinant vector containing thepolynucleotide according to claim
 39. 42. The recombinant vectoraccording to claim 40, wherein the recombinant vector is a recombinantexpression vector.
 43. A transformant that is transformed with therecombinant vector according to claim
 42. 44. A method for producing thepeptide according to claim 2, wherein the method comprises a step ofincubating a transformant transformed with a recombinant expressionvector comprising a nucleotide sequence that encodes the peptide.
 44. Anantibody that immunologically recognizes the peptide according to claim2.
 45. An antibody that immunologically recognizes the peptide accordingto claim 2 and that inhibits the binding of the peptide to a proteinthat interacts with the peptide.
 46. An antibody that immunologicallyrecognizes the peptide according to claim 2 and that inhibits thebinding of the peptide to human syntaxin-1a.
 47. A method foridentifying a compound that inhibits or stabilizes the binding of thepeptide according to claim 2 to a protein that interacts with thepeptide, wherein the method comprises steps of: contacting a testsubstance with the peptide and the protein that interacts with thepeptide, under conditions that allow the binding of the peptide to theprotein that interacts with the peptide; and determining whether thetest substance inhibits or stabilizes the binding of the peptide to theprotein that interacts with the peptide, by detecting the presence,absence, or change of a signal generated by the binding of the peptideto the protein that interacts with the peptide.
 48. A method foridentifying a compound that inhibits or stabilizes the binding of thepeptide according to claim 2 to human syntaxin-1a, wherein the methodcomprises steps of: contacting a test substance with the peptide andhuman syntaxin-1a, under conditions that allow the binding of thepeptide to human syntaxin-1a; and determining whether the test substanceinhibits or stabilizes the binding of the peptide to human syntaxin-1a,by detecting the presence, absence, or change of a signal generated bythe binding of the polypeptide or the peptide to human syntaxin-1a. 49.A method for identifying a compound that inhibits or promotes expressionof the polynucleotide according to 38, wherein the method comprisessteps of: contacting a test substance with the polynucleotide underconditions that allow expression of the polynucleotide; and determiningwhether the test substance inhibits or promotes expression of thepolynucleotide by detecting the presence, absence, or change of a signalgenerated by expression of the polynucleotide.
 50. A compound that isidentified by the method according to claim
 16. 51. A compound that isidentified by the method according to claim
 17. 52. A compound that isidentified by the method according to claim
 18. 53. A compound that isidentified by the method according to claim
 51. 54. A compound that isidentified by the method according to claim
 52. 55. A compound that isidentified by the method according of claim
 53. 56. A compound thatinhibits or stabilizes the binding of the peptide according to claim 2to human syntaxin-1a by interacting with the peptide.
 57. A compoundthat inhibits or promotes expression of the polynucleotide according toclaim 7 by interacting with the polynucleotide.
 58. An agent forpromoting the formation of a complex that comprises human syntaxin-1a,synaptosomal-associated 25 kDa protein and synaptotagmin andparticipates in fusion of a synaptic vesicle with a presynapticmembrane, wherein the agent comprises the peptide according to claim 2.59. An inhibitor comprising an antibody that immunologically recognizesthe peptide according to claim 2 and that inhibits the binding of thepeptide according to claim 2 to a protein that interacts with thepeptide.
 63. An inhibitor comprising an antibody that immunologicallyrecognizes the peptide according to claim 2 and that inhibits thebinding of the peptide according to claim 2 to human syntaxin-1a.